Ribosomal pausing and scanning arrest as mechanisms of translational regulation from cap-distal iron-responsive elements.

Iron regulatory protein 1 (IRP-1) binding to an iron-responsive element (IRE) located close to the cap structure of mRNAs represses translation by precluding the recruitment of the small ribosomal subunit to these mRNAs. This mechanism is position dependent; reporter mRNAs bearing IREs located further downstream exhibit diminished translational control in transfected mammalian cells. To investigate the underlying mechanism, we have recapitulated this position effect in a rabbit reticulocyte cell-free translation system. We show that the recruitment of the 43S preinitiation complex to the mRNA is unaffected when IRP-1 is bound to a cap-distal IRE. Following 43S complex recruitment, the translation initiation apparatus appears to stall, before linearly progressing to the initiation codon. The slow passive dissociation rate of IRP-1 from the cap-distal IRE suggests that the mammalian translation apparatus plays an active role in overcoming the cap-distal IRE-IRP-1 complex. In contrast, cap-distal IRE-IRP-1 complexes efficiently repress translation in wheat germ and yeast translation extracts. Since inhibition occurs subsequent to 43S complex recruitment, an efficient arrest of productive scanning may represent a second mechanism by which RNA-protein interactions within the 5' untranslated region of an mRNA can regulate translation. In contrast to initiating ribosomes, elongating ribosomes from mammal, plant, and yeast cells are unaffected by IRE-IRP-1 complexes positioned within the open reading frame. These data shed light on a characteristic aspect of the IRE-IRP regulatory system and uncover properties of the initiation and elongation translation apparatus of eukaryotic cells.

Expression of the human homologue of rat NG2 in adult acute lymphoblastic leukemia: close association with MLL rearrangement and a CD10(-)/CD24(-)/CD65s(+)/CD15(+) B-cell phenotype.

The expression of the chondroitin sulfate proteoglycan neuron-glial antigen 2 (NG2) has been demonstrated in association with rearrangement of the mixed lineage leukemia (MLL) gene in acute leukemia, but the frequency of NG2 expression in adult acute lymphoblastic leukemia (ALL) is yet unknown. We evaluated NG2 expression in 313 adult ALL patients by flow cytometry and simultaneously determined MLL rearrangement in 120 adult patients out of them with B-precursor ALL by reverse transcription-polymerase chain reaction and fluorescence in situ hybridization. A total of 57% of pro-B ALL, 2% of common ALL and 20% of pre-B ALL were NG2 positive, but NG2 was absent in T-ALL and mature B-ALL. In B-precursor ALL, NG2 expression was significantly associated with a CD10(-)/CD34(-)/CD24(-)/CD65s(+)/CD15(+)/CD13(-)/CD33(-) phenotype and showed a sensitivity, specificity and positive predictive value of 0.89, 0.89, and 0.93 for MLL rearrangement, respectively. NG2 was positive in three patients without detectable MLL rearrangement and negative in eight patients with MLL-AF4 transcripts. However, NG2 predicted with a 100% accuracy MLL rearrangement among patients disclosing a CD65s(+) and/or CD15(+) immunophenotype. In summary, NG2 adds to a more precise identification of high-risk adult ALL and should therefore be included into diagnostic marker panels. As NG2 is negative in non-malignant hematopoietic cells, this novel antigen might also serve in future studies as a powerful marker in monitoring minimal residual disease.

Evaluation of a cassette-screen-film combination for radiation therapy portal localization imaging with improved contrast.

A traditional limitation with radiation therapy portal images is low image contrast, due in part to the low attenuation of the exposing radiation by the tissues being imaged, and the contrast capabilities of the image receptor. We have developed, and have clinically evaluated, a cassette-screen-film combination for portal localization imaging, which features a copper front screen plus Gd2O2S:Tb fluorescent screens and a slow-speed, fine grain, film emulsion with inherently high contrast coated on both sides of a 7 mil Estar base. The film can be processed in a conventional rapid-process film processor. Sensitometric data indicate that the film contrast (average gradient) for the new combination is approximately 3.5 times higher than the conventional portal localization systems in current use. The new combination has been clinically compared with two conventional systems. The required monitor unit settings were found to be similar. Initial clinical results indicate portal images made with the new combination are superior to those obtained with the conventional combinations. The images have much higher contrast, subjective impressions of lower noise, show clearer definition of structures, and are much easier to read.

[CO2-stunning of swine for slaughter from the anesthesiological viewpoint]. / Die CO2-Betäubung von Schlachtschweinen aus anästhesiologischer Sicht.

For investigations of CO2-stunning of feeder- and slaughter-pigs parameters of behaviour, blood-gas-analyses and electroencephalograms were chosen. The following results were obtained: 1. Blood-gas-analyses proved that the CO2-stunning does not produce unconsciousness due to a lack of oxygen. 2. The criterias of general anaesthesia: unconsciousness, muscle-relaxation and analgesia with total reversibility could be confirmed. 3. The violent convulsive symptoms were evaluated as reactions identical with the stage II of GUEDEL's scheme of anaesthesia. 4. Muscular agitation, which sometimes appeared a few seconds before the stage of excitation, was judged to belong either to the start of the excitation phase or to the end of Guedel's stage of analgesia, during which the sensitivity is decreased. Neither study of behavior nor objective measurements showed, during the first 10 to 20 seconds of exposure to the CO2, any sign of pain or suffering related to the Act for Prevention of Cruelty to Animals, and accordingly such suffering should not be ascribed to the CO2 stunning method.

[Secondary forearm deformity due to premature closure of the distal ulnar physis]. / Posttraumatische Deformität am distalen Unterarm nach vorzeitigem Verschluss der ulnaren Wachstumsfuge.

We report on a 16-year-old boy with deformity of the forearm with painful functional limitation after a fracture of the distal radius associated with an unapparent lesion of the distal physis of the ulna suffered 4 years earlier. The lesion caused premature growth arrest in the ulna and a bowing of the distal radius with a carpal slip. It was treated with a corrective osteotomy in the radius together with a lengthening of the ulna, with excellent functional results.

[Recurrent laryngeal nerve paralysis after thyroidectomy]. / Rekurrensparesen nach Strumektomien.

AIM: The paper's aim is to examine and analyse the incidence rate of postoperative pareses of N. recurrens in 1311 operated thyroid sides and 909 patients. PATIENTS AND SURGICAL TECHNIQUE: In histology we found nodular changes in 92.6% of 840 operated benignant strumata. In 69 malignant strumata the papillary thyroid carcinoma was the most common. The surgical concept changed within the last years from subtotal resections and enuclations to functional and total resections. RESULTS: Preoperative pareses of N. recurrens were seen in 5.3% of the patients, which emphasizes the demand for a preoperative laryngologic examination. Surgery on relapsed benignant strumata were followed by permanent unilateral pareses of N. recurrens in 9.9%, surgery on malignant strumata in 7.5% of the cases. After surgery of relapsed malignant strumata we found 2 permanent pareses in 21 reoperated thyroid sides. During a regular follow-up over the period of 12 months at least, we saw 22 transitory pareses of N. recurrens, which corresponds to a reversibility rate of 35%. In first surgical interventions with benignant histology the reversibility rate was 49%. Depending on first or relapse surgery, the histological findings and chosen surgical method the rate of permanent unilateral pareses of N. recurrens was only 1.7% in relation to the first time operated sides of benignant strumata.

Ca(2+)-activated K+ channels in human smooth muscle cells of coronary atherosclerotic plaques and coronary media segments.

The behavior of Ca(2+)-activated K+ channels of large conductance (BKCa) in smooth muscle cells, which were obtained from atherosclerotic plaque material (SMCP) and from media segments (SMCM) of human coronary arteries, were compared using the patch-clamp technique. Voltage-clamp protocols in cell-attached patches revealed the characteristic voltage-dependent activation of BKCa in both cell groups. Single-channel conduction as 216.4 +/- 16.7 pS (n = 6) in SMCP and 199.9 +/- 6.7 pS (n = 6) in SMCM in symmetrical 140 mM K+ solutions. Using outside-out patches, external perfusion with 500 microM tetraethylammonium ions caused a typical "flickery block" of the unitary current. The selective BKCa channel inhibitor iberiotoxin (50 nM) effectively blocked BKCa, channel activity. Comparing BKCa open-state probabilities (P0) at +80 mV in cell-attached patches, a highly significant difference between SMCP (P0 = 0.1438 +/- 0.1301; n = 15) and SMCM (P0 = 0.0093 +/- 0.0044; n = 15; Kruskal-Wallis test, p < 0.001) was found. In contrast to this finding, there was no significant difference in the open-state probability of BKCa, between SMCP (P0 = 0.542 +/- 0.0237; n = 9) and SMCM (P0 = 0.0472 +/- 0.0218; n = 10; p = n.s.) using inside-out patches. The results show an interesting difference in the behavior of large conductance Ca(2+)-activated K+ channel in SMCP compared to SMCM with a significantly higher channel activity in human smooth muscle cells obtained from coronary atherosclerotic plaque material. This finding may indicate an important functional role of BKCa channels in the development of atherosclerosis.

Ligand-induced structural alterations in human iron regulatory protein-1 revealed by protein footprinting.

Human iron regulatory protein-1 (IRP-1) is a bifunctional protein that regulates iron metabolism by binding to mRNAs encoding proteins involved in iron uptake, storage, and utilization. Intracellular iron accumulation regulates IRP-1 function by promoting the assembly of an iron-sulfur cluster, conferring aconitase activity to IRP-1, and hindering RNA binding. Using protein footprinting, we have studied the structure of the two functional forms of IRP-1 and have mapped the surface of the iron-responsive element (IRE) binding site. Binding of the ferritin IRE or of the minimal regulatory region of transferrin receptor mRNA induced strong protections against proteolysis in the region spanning amino acids 80 to 187, which are located in the putative cleft thought to be involved in RNA binding. In addition, IRE-induced protections were also found in the C-terminal domain at Arg-721 and Arg-728. These data implicate a bipartite IRE binding site located in the putative cleft of IRP-1. The aconitase form of IRP-1 adopts a more compact structure because strong reductions of cleavage were detected in two defined areas encompassing residues 149 to 187 and 721 to 735. Thus both ligands of apo-IRP-1, the IRE and the 4Fe-4S cluster, induce distinct but overlapping alterations in protease accessibility. These data provide evidences for structural changes in IRP-1 upon cluster formation that affect the accessibility of residues constituting the RNA binding site.

Probing the structure of the regulatory region of human transferrin receptor messenger RNA and its interaction with iron regulatory protein-1.

A portion of the 3'UTR of the human transferrin receptor mRNA mediates iron-dependent regulation of mRNA stability. The minimal RNA regulatory region contains three conserved hairpins, so-called iron responsive elements (IREs), that are recognized specifically by iron regulatory proteins (IRPs). The structure of this regulatory region and its complex with IRP-1 was probed using a combination of enzymes and chemicals. The data support the existence of an intrinsic IRE loop structure that is constrained by an internal C-G base pair. This particular structure is one of the determinants required for optimal IRP binding. IRP-1 covers one helical turn of the IRE and protects conserved residues in each of the three IREs: the bulged cytosine and nucleotides in the hairpin loops. Two essential IRP-phosphate contacts were identified by ethylation interference. Three-dimensional modeling of one IRE reveals that IRP-1 contacts several bases and the ribose-phosphate backbone located on one face in the deep groove, but contacts also exist with the shallow groove. A conformational change of the IRE loop mediated by IRP-1 binding was visualized by Pb2+-catalyzed hydrolysis. This effect is dependent on the loop structure and on the nature of the closing base pair. Within the regulatory region of transferrin receptor mRNA, IRP-1 induces reactivity changes in a U-rich hairpin loop that requires the presence of the stem-loop structure located just downstream the endonucleolytic cleavage site identified by Binder et al. (Binder R et al. 1994, EMBO J 13:1969-1980). These results provide indications of the mechanism by which IRP-1 stabilizes the transferrin receptor mRNA under iron depletion conditions.